Splenic lymphocytes from young, 15-month-old and 24-month-old C57BL/6 mice housed in a constant environment were studied. Single cell suspensions, both unfrozen and cryopreserved, were cultured in vitro with the T cell mitogens, phytohaemagglutinin and Concanavalin A, and the B cell mitogen, lipopolysaccharide. Functional capacity of the T and B lymphocytes was assessed by the mitogen-induced incorporation of tritiated thymidine by dividing cells. Seasonal rhythmicity in the incorporation of tritiated thymidine was exhibited by splenic lymphocytes from mice of three different ages. The responses to both T-cell mitogens exhibited marked decreases in amplitude and increase in freerunning periods with age. Activated B-cells exhibited little change in amplitude but markedly longer freerunning periods. In comparing the three age groups, the rhythms were not in synchrony with each other, and those of the older mice were not in synchrony with the calendar year. The "variability" and lack of age-related declines in lymphocytic responses to mitogens previously reported by others may be due to grouping data for all seasons and to testing during phases of the circannual rhythm characterized by similar responses of all age groups. Cryopreservation of lymphocytes from older mice resulted in lower recoveries of viable and functional cells as compared to cryopreserved lymphocytes from young mice. Seasonal rhythmicities in the recoveries of young mouse cells also were exhibited and paralleled those of unfrozen cells. The significance of these results is 1) their support of the hypothesis that decay of circadian and circannual organization may be involved in physiological deterioration with age, and 2) cryopreservation stress confers selective injury, probably involving the plasma membrane, to lymphocytes from older mice.